Apoptosis is a normal physiological process of cell death that plays a critical role in the regulation of tissue homeostasis by ensuring that the rate of new cell accumulation produced by cell division is offset by a commensurate rate of cell loss due to death. It has now become clear that disturbances in apoptosis, also referred to as physiological cell death or programmed cell death, that prevent or delay normal cell turnover can be just as important to the pathogenesis of diseases as are known abnormalities in the regulation of proliferation and the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death.
The stimuli which regulate the function of these apoptotic gene products include both extracellular and intracellular signals. Either the presence or the removal of a particular stimuli can be sufficient to evoke a positive or negative apoptotic signal. For example, physiological stimuli that prevent or inhibit apoptosis include, for example, growth factors, extracellular matrix, CD40 ligand, viral gene products, neutral amino acids, zinc, estrogen, and androgens. In contrast, stimuli which promote apoptosis include growth factors such as tumor necrosis factor (TNF), Fas, and transforming growth factor xcex2 (TGFxcex2), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment, intracellular calcium and glucocorticoids, for example.
Some of the well known regulators of apoptosis are cytokines of the tumor necrosis factor (TNF) ligand family, such as Fas ligand (Fas L) and TNF, which induce apoptosis by activation of their corresponding receptors, Fas and TNFR-1 (Nagata, S. (1997) Cell 88, 355-36S). These two receptors belong to a rapidly expanding family (collectively known as the TNF-receptor family) containing at least eleven known members (Nagata, S. (1997) Cell 88, 355-365; Chinnaiyan, A. M. et al. (1997) xcexa3"khgr"lxcex5xcexd"khgr"xcex5 6, 111-113). Members of this family contain an extracellular ligand-binding domain, of 2-6 cysteine-rich repeats, which is about 25% conserved between different family members. The cytoplasmic region is less conserved between various members except for a stretch of about 80 amino acids present in Fas, TNFR-1, DR3/Wsl-1/Apo-3/TRAMP, CAR-1 and DR4 (Nagata, S. (1997) Cell 88, 355-365; and references therein). This intracellular region which has been designated the cytoplasmic xe2x80x9cdeath domainxe2x80x9d is responsible for transducing the death signal.
Activation of Fas results in recruitment of the Fas-associated death domain-containing molecule FADD/MORT-1, to the receptor complex (Boldin M. P. et al. (1995) J. Biol. Chem. 270, 7795-7798; Chinnaiyan A. M. et al. (1995) Cell 81, 505-512; Kischkel F. C. (1995) EMBO J. 14, 5579-5588). The resulting signaling complex then triggers activation of the caspase apoptotic pathway through interaction of the N-terminal death effector domain (DED) of FADD with the corresponding motifs in the prodomain of caspase-8 (Mch5/MACH/FLICE) and probably caspase-10 (Mch4) (Boldin M. P. et al. (1996) Cell 85, 803-815; Bretz J. D. et al. (1996) Cell 85, 817-827; Alnemri E. S. et al. (1996) Proc. Natl. Sci. USA. 93, 7464-7469; Alnemri E. S. et al. (1996) Cell 87, 171.
In contrast to Fas, activation of TNFR-1 or DR3 results in recruitment of another death domain-containing adaptor molecule known as TRAD (Chinnaiyan A. M. et al. (1996) Science 274, 990-991; Goeddel D. V. et al. (1995) Cell 81: 495-504). TRADD, can associate with a number of signaling molecules, including FADD, TRAF2, and RIP and as a result can transduce an apoptotic signal as well as activate NF-kB (Goeddel D. V. et al. (1996) Cell 84, 299-308; Baichwal V. et al. (1996) Immunity 4, 387-396). Consequently, engagement of TNFR-1 or DR3 can signal an array of diverse biological activities.
Recently, a new member of the TNF family known as TRAIL or Apo-2 ligand was identified and shown to induce apoptosis in a variety of tumor cell lines (Davis T. D. et al. (1995) Immunity 3 673-682: Ashkenazi A. et al. (1996) J. Biol. Chem. 271, 12687-12690; Ashkenazi A. et al. (1996) Curr. Biol. 6, 750-752). However, it is unclear what physiological control mechanisms regulate this form of programmed cell death or how the cell death pathways interact with other physiological processes within the organism.
Apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover. Therefore, the dysfunction, or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes such as that occurring with many autoimmune diseases. Inappropriate loss of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such an neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can change the natural progression of many of these diseases.
Thus, there exists a need to identify new apoptotic genes and their gene products and for methods of modulating this process for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
In accordance with the present invention, there are provided novel isolated mammalian members of the TRAIL-receptor family, designated DR5, TRAIL-R3, and splice variants thereof including DR5s. These invention proteins, or fragments thereof, are useful as immunogens for producing anti-DR5 or anti-TRAIL-R3 antibodies, or in therapeutic compositions containing such proteins and/or antibodies. The DR5 and TRAIL-R3 proteins are also useful in bioassays to identify agonists and antagonists thereto.
In accordance with the present invention, there are also provided isolated nucleic acids encoding novel DR5 or TRAIL-R3 proteins. Further provided are vectors containing invention nucleic acids, probes that hybridize thereto, host cells transformed therewith, antisense oligonucleotides thereto and related compositions. The nucleic acid molecules described herein can be incorporated into a variety of recombinant expression systems known to those of skill in the art to readily produce isolated recombinant DR5 or TRAIL-R3 proteins. In addition, the nucleic acid molecules of the present invention are useful as probes for assaying for the presence and/or amount of a DR5 or TRAIL-R3 gene or mRNA transcript in a given sample. The nucleic acid molecules described herein, and oligonucleotide fragments thereof, are also useful as primers and/or templates in a PCR reaction for amplifying nucleic acids encoding DR5 or TRAIL-R3 proteins. Also provided are transgenic non-human mammals that express the invention proteins.
Antibodies that are immunoreactive with invention DR5 or TRAIL-R3 proteins are also provided. These antibodies are useful in diagnostic assays to determine levels of DR5 or TRAIL-R3 proteins present in a given sample, e.g., tissue samples, Western blots, and the like. The antibodies can also be used to purify DR5 or TRAIL-R3 proteins from crude cell extracts and the like. Moreover, these antibodies are considered therapeutically useful to modulate the biological effect of DR5 or TRAIL-R3 proteins in vivo.
Methods and diagnostic systems for determining the levels of DR5 or TRAIL-R3 proteins in various tissue samples are also provided. These diagnostic methods can be used for monitoring the level of therapeutically administered DR5 or TRAIL-R3 proteins or fragments thereof to facilitate the maintenance of therapeutically effective amounts. These diagnostic methods can also be used to diagnose physiological disorders that result from abnormal levels or abnormal structures of the DR5 or TRAIL-R3 proteins.